Файл: Extracellular and Intracellular Signaling (книга).pdf

have been inspired by these experiments and starting from inactive structures (from crystals) provided a more detailed view of these ligand-stabilized conformations,61,62 which is consistent with experimental observations. These observations of ligand-stabilized conformational states of a GPCR open the possibility of di erent agonists stabilizing di erent active conformations with di erent functional roles as will be seen later.

The seven-helix topology of GPCRs also enables a mutation to have a significant e ect on their conformations accessible under physiological conditions. The Kendall group was able to show using two mutations at the same position in the cannabinoid CB1 receptor that the receptor can be switched to the constitutively active form (R*) or into the inactive form (R) with no basal activity.52 They found that the T210I mutant converts the CB1 receptor into a more fully constitutively active state (R*), showing enhanced binding e cacy for agonists and diminished binding e cacy for inverse agonists. They also find that the T210A mutant is constitutively inactive, i.e. it converts the CB1 receptor into a fully inactive state (R), which shows enhanced binding e cacy for inverse agonists and weaker binding for agonists. This strongly suggests that even in the absence of any ligand, GPCRs are capable of major conformational changes with potentially different functional outcomes.

We have recently predicted an ensemble of structures (conformations) for the CCR5 chemokine receptor that is a co-receptor for HIV entry using our GEnSeMBLE method for the e cient optimization of helix tilt angles (y, f) and rotation angle (Z) that were defined in Figure 11.2A. The top lowest energy predicted conformations are shown in Table 11.2 and how they di er from each other in terms of structure and energies.

We have also discovered that mutations can have significant impact on the ensemble of conformations accessible by the apo proteins. Table 11.3 and Figure 11.8 illustrate this using CCR5 mutants as examples. The W86A CCR5 mutant conformations show that the wild-type #30 (wt30) structure now ranks at #4 (Table 11.3). Also, Figure 11.9 shows that this Trp86 residue has a steric clash with helix 7 in the wild-type structure, which disappears in the W86A mutant increasing its stability. The table also shows that for the A90H mutant, the wt1 structure disappears from the top conformations, in this case due to steric clash of His90 with helix 7.

This phenomenon is not fully appreciated for GPCRs, but has the potential to explain the origin of the e ect of mutations on ligand binding, whether the mutation a ected the protein structure or directly a ected ligand binding. We have docked multiple ligands to wild-type CCR5 and mutant ensemble of conformations. The results for binding of Maraviroc (the only drug in the market aimed at CCR5) are shown in Table 11.4, which shows the di erent protein conformations preferred by the wt and mutant proteins in apo and Maraviroc bound forms. We also find that di erent ligands (Maraviroc, PF-232798, Aplaviroc) prefer di erent CCR5 conformations, which is consistent with observations that these ligands bind to the same site but show di erent interaction profiles in mutagenesis studies63,64 and in e ects on antibody binding.65

Table 11.3 Reordering of CCR5 wild-type conformations for mutants W86A, A90H and T105A.

Figure 11.9 Structural basis for mutation e ect on conformations (see text).

Next, we will describe the known intracellular consequences of GPCR activation and how the sensing of a single signal by the receptor can turn on a very wide range of intracellular signaling cascades.

Table 11.4 Reordering of wild-type CCR5 conformations for various mutants and the conformation preferred by Maraviroc for each of the mutants.

11.3.2.3Intracellular Signal Diversification and GPCR Regulation

GPCR activation can lead to a diverse set of signaling events inside the cell. So, these receptors can be called pleiotropic. These signaling events are mainly transduced through multiple G proteins and arrestins (Figure 11.10). GPCR coupling to G proteins (which gave these receptors the name ‘‘GPCR’’) enables signal diversification inside the cell as these G proteins control a spectrum of downstream signaling pathways. These pathways respond within seconds to minutes of the sensing of the signal by the GPCR. Arrestins appear to be playing a dual role of signal diversification as well as GPCR regulation (receptor desensitization and internalization). Arrestin-mediated signaling responses have a slower onset and are sustained over minutes to hours. As will be shown later, the ability of a single receptor to activate multiple pathways inside the cell can have important therapeutic consequences, as the G proteinmediated pathway may have a therapeutic benefit and the arrestin-mediated pathway may have unintended side-e ects or vice versa. Next we will briefly describe the G protein-mediated and arrestin-mediated pathways and how their actions define GPCR function.

11.3.2.3.1 G Protein Mediated Signal Diversification. GPCRs got their name due to coupling to G proteins, which are heterotrimeric proteins with

A

B

Figure 11.10 A G protein and arrestin-dependent signaling pathways. B Di erence in response times from di erent pathways. Reproduced with permission from American Society for Pharmacology and Experimental Therapeutics from Luttrell and Gesty-Palmer, Pharmacol. Rev., 62, 305–330. Copyright 2010.

Ga, Gb and Gg subunits. The Ga subunit is made up of a ras-like GTPase domain and a helical domain. The guanine nucleotide GDP is sandwiched between these two domains. There are 23 known Ga proteins that can be

assigned to four major classes represented by Gas, Gai/o, Gaq/11 and Ga12/13 proteins. The Gb subunit forms a bridge between Ga and Gg subunits,

which don’t appear to have a close contact as shown in Figure 11.11. There are 5 known human Gb and 12 human Gg subunits, which can lead to a large number of possible Gbg dimers that are coupled to many downstream signaling pathways66,67 as will be discussed below.

The inactive G protein exists in the heterotrimeric form bound to ADP. The activation of a GPCR upon agonist binding leads to a series of conformational

(A)

(B)

Figure 11.11 The architecture of the heterotrimeric G protein.

changes, which recruit binding to the G protein using the receptor’s intracellular loops and the C-terminus residues interacting mainly with the Ga subunit. This association leads to the dissociation of the G protein into the Ga subunit (which undergoes a conformational change coupled to the displacement of ADP by ATP) and the heterodimer complex Gbg as shown in the left panel of Figure 11.12 that doesn’t undergo any major conformational change. Each of these units, the Ga subunit and the Gbg complex, activates specific signaling pathways inside the cell. A recent crystal structure of Opsin (ligand-free form of rhodopsin) bound to the carboxy terminus of the Ga subunit28 has shed light on the detailed molecular level contacts present between GPCRs and G proteins as shown in the right panels of Figure 11.12, which highlights through the middle panel the conformational change necessary upon activation to accommodate binding to the Ga subunit.

The Ga subunit controls many downstream signaling cascades through di erent e ector molecules. The Gas class of proteins stimulate adenylyl cyclase (AC), which converts ATP into cyclic AMP (cAMP), whereas Gai class inhibits AC thereby opposing the e ect of Gas class. This suggests how a single GPCR activated by the same agonist can have opposite e ects inside the cell just by coupling to di erent G proteins. The Gaq class of subunits activate phospho-inositide-specific phospholipase C (PI-PLC) isoenzymes, which generate second messengers inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 activates Ca21 channels leading to the increase in calcium concentration, which facilitates protein kinase C (PKC) activation by DAG. Besides this critical function that controls many cellular functions through phosphorylation events caused by PKC, DAG also acts as a source of prostaglandins and as a precursor of the endocannabinoid 2-arachido- noylglycerol among its other functions.

The Gbg complex activates its own set of e ectors and the first ones identified were G protein-regulated inward-rectifier K1 channels (GIRKs).

pathway, or via di erent e ectors leading to multiple signaling pathways (Figure 11.13B).

c)Agonist binding to GPCR activates a G protein leading to a single signaling pathway inside the cell, which diverges downstream (Figure

11.13C).

d) GPCR activation allows it to couple to multiple G proteins inside the cell, which can activate many signaling pathways (Figure 11.13D).

These di erent scenarios are enabled due to conformational flexibility of activated GPCRs, which allows them a range of controls over intracellular signaling pathways. Next, we will describe the interaction of GPCRs with arrestins, which is another mode by which they activate various pathways inside the cell.

11.3.2.3.2 Arrestin Mediated Signaling and GPCR Regulation. b-Arrestins were originally thought to be only responsible for desensitization of activated GPCRs followed by internalization and recycling or degradation. As will be shown below, this view has changed dramatically and arrestins can activate multiple signaling pathways independent of any G protein induced signaling. The arrestin family comprises four proteins in vertebrates: visual arrestins (arrestins 1 and 4) as well as b-arrestins 1 and 2 (arrestins 2 and 3).68

Desensitization of GPCRs (or loss of G protein-coupled response in the presence of an agonist) has been reviewed elsewhere70 and lucidly depicted (along with the endocytosis mechanisms) in Figure 11.14. A succinct picture of classical desensitization involves receptor phosphorylation by second messen- ger-stimulated protein kinases (like PKA and PKC) or by specific GRKs followed by arrestin binding to the intracellular receptor site that sterically hinders any further G protein coupling. In addition, two new roles have emerged for b-arrestins in desensitization: a) degradation of second messenger molecules like cAMP71 and diacylglycerol (DAG);72 and b) limiting or reducing the generation of second messenger molecules as shown in a study73 that b-arrestin promotes the switching from Gs to Gi coupling, which further limits the production of cAMP. The class A GPCRs show greater a nity for b-arrestin 2, whereas class B GPCRs bind to b-arrestin 1 and 2 with equal a nity.

The receptor internalization (or endocytosis) is mediated by b-arrestin binding to clathrin via the adaptor protein AP-2, which targets the GPCRs to clathrin-coated pits (CCPs). These CCPs are pinched o from the plasma membrane by a large GTPase dynamin (see Figure 11.14) into endosomal vesicles, which either recycle the receptor to the plasma membrane or take the receptor through degradation pathways. In the vesicle, the agonists are released from the receptor due to the acidic environment of the vesicle and the receptor is dephosphorylated in preparation for recycling back to the plasma membrane (or resensitization).

One of the important functions of GPCRs is the regulation of cell proliferation and di erentiation, via the activation of mitogenic pathways. The